Urinary incontinence is a major health problem in the United States, resulting in an 11% lifetime risk of surgery for women with incontinence and/or prolapse. The molecular pathophysiology is poorly understood, inhibiting the development of medical prophylaxis or therapy for this growing area of disability. Collagen and elastin are major supporting elements of pelvic structures. Thus, expression of collagenases, elastases and their inhibitors controls proteolysis of the extracellular matrix and may negatively impact urinary function. We hypothesize that stress urinary incontinence (SUI) results from abnormal pelvic collagen and elastin metabolism as a function of differential expression of the matrix metalloproteinases (MMPs) and elastases and their respective protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) and A-1 anti-trypsin. First, we propose to investigate expression of both the proteolytic enzymes and their inhibitors in vaginal wall tissue isolated from women with SUI and continent women during the follicular and luteal phases of the menstrual cycle using quantitative-competitive reverse transcription polymerase chain reaction (QC-RT-PCR), Western blot, immunohistochemical analysis, and zymography. Second, we will compare the expression of MMPs and TIMPs as well as neutrophil elastase and A-1 anti-trypsin in vaginal tissue samples from continent and incontinent postmenopausal women to determine the effect of estrogen deprivation on RNA and protein expression as well as on collagen and elastin proteolytic activity. In addition, we will compare broad patterns of gene expression between continent and incontinent women using microarray analysis. Third, to assess the role of growth factors in the etiology of urinary dysfunction, we will examine relaxin and transforming growth factor-B (TGF-B) modulation of both collagenolysis and elastolysis in cultured fibroblasts derived from premenopausal continent and incontinent women. Because urinary dysfunction develops as women age, our fourth goal is to examine the ability of 17-B estradiol and progesterone to alter in vitro expression of the ratio of MMPs/TIMPs and total elastase activity/A-1 anti-trypsin in vaginal fibroblast cultures. Two-thirds of the burden of urinary incontinence is borne by women with prevalence rates of 14-41%. Our goal is to identify specific pathophysiologic changes related to reproductive events and aging which underlie the development of SUI as a first step to identifying potential targets for therapeutic intervention.